The long-term objective of the applicant's research program is to understand regulatory mechanisms responsible for controlling mitochondrial fatty acid metabolism. A two-year project is proposed to investigate acetylation of lysine residues on members of the acyl-CoA dehydrogenase (ACAD) family of proteins. ACADs are mitochondrial flavoenzymes that catalyze the first step in fatty acid oxidation. Genetic deficiencies of these enzymes are among the most common inborn errors of metabolism. Additionally, obesity, diabetes, and other common diseases have been linked to dysfunctional mitochondrial fatty acid oxidation. Because they catalyze the rate-limiting step in the fatty acid oxidation cycle, factors that regulate ACADs regulate the rate of energy production from fatty acids. Preliminary data demonstrate that post-translational modification by lysine acetylation can regulate activity of long- chain acyl-CoA dehydrogenase (LCAD) by two-fold. The mechanism by which LCAD and other mitochondrial proteins become acetylated is not known. While lysine acetyltransferases are present in the nucleus and other intracellular compartments, none have been identified in mitochondria. Preliminary data show that acetylation of several ACAD enzymes can occur chemically. Incubation of purified recombinant ACADs with acetyl-CoA at concentrations greater than 0.5 mM resulted in lysine acetylation. Since mitochondrial acetyl-CoA concentrations may reach 2 mM or higher it is hypothesized that chemical acetylation is responsible for the phenomenon of lysine acetylation seen in mitochondria of highly oxidative tissues such as liver, muscle, heart and brown fat. Two aims are proposed to study the phenomenon. Aim 1 is to characterize non-enzymatic lysine acetylation of ACAD enzymes. This includes a comprehensive survey of reaction conditions, the use of acetyltransferase inhibitors, and multiple methods of detecting and quantifying lysine acetylation. Aim 2 is to determine how microenvironments on the surface of ACAD proteins influence lysine acetylation. Based on three-dimensional molecular modeling and other preliminary data it is postulated that lysines located near acidic amino acids are particularly prone to modification due to active deprotonation by the acidic residues. Recombinant ACADs acetylated by reaction with acetyl-CoA will be subjected to mass spectrometry to identify the acetylation sites. These sites will then be mapped in three-dimensional space using molecular modeling software and previously published ACAD crystal structures. Finally, site-directed mutagenesis will be used to determine the contribution of neighboring amino acids to the acetylation of each identified lysine. The proposed R03 project would synergize with the applicant's current K01 Career Development Award resulting in a more rapid transition to independence and a more competitive future R01 application. PUBLIC HEALTH RELEVANCE: Public Health Relevance Changes in mitochondrial energy metabolism underlie many major diseases from cancer to diabetes, and are also believed to be at the core of the aging process. The proposed research seeks to elucidate the molecular events behind a modification to metabolic enzymes known as lysine acetylation. Understanding lysine acetylation of mitochondrial enzymes may lead to the development of new drugs to control metabolism, thereby preventing disease and perhaps even slowing aging.